The method of obtaining a strain of bacteriofage, specific strains of bacteriophage and use thereof

ABSTRACT

A method for obtaining a strain of bacteriophage specific to a selected strain of bacteria was found as well as bacteriophage strains obtained in this way. Moreover, application of bacteriophages in manufacturing of the preparation for preventing and fighting infections of farm animals, especially poultry, with pathogenic strains of bacteria sensitive to these phages was described.

This invention relates to a method for preparing a strain ofbacteriophage specific to a selected strain of bacteria, bacteriophagestrains obtained in this way and the application of bacteriophages tomanufacture preparation for preventing and fighting infections of farmanimals, especially poultry, with pathogenic strains of bacteriasensitive to these bacteriophages.

The aim of the invention is to provide production technology for anantimicrobial preparation suitable for use as a feed additive forpoultry and pigs, which at the same time would be specific to pathogenicstrains of Salmonella spp that cause incidence of salmonellosis,especially in humans. The preparation shall comply with strict safetyrequirements for feed additives. Prohibition to use antibiotics inanimal feed, valid in European Union countries since 1^(st) January2006, created a huge demand for feed additives, not containingantibiotics, but having anti-microbial effect.

The aim of the present invention is to provide a preparation that couldreplace the currently used antibiotics.

Unexpectedly, such preparation was successfully obtained in the presentinvention.

This invention relates to a method of preparing a strain ofbacteriophage specific to a selected strain of bacteria, characterizedin that:

-   -   a) a collection of bacteriophage strains containing a strain of        bacteriophage specific to a selected strain of bacteria is        obtained,    -   b) culture of a selected strain of bacteria on a sterile culture        medium is conducted,    -   c) the culture samples are applied on the special multi-well        measuring plate, then a suspension of tested bacteriophage        strain at various concentrations is added and it is incubated at        37° C. for at least 4 hours,    -   d) resazurin is added to the culture and the incubation is        continued in the dark at 37° C., for at least 3 hours,    -   e) the colour or fluorescence of culture is being controlled, as        well as bacteriophage strain contained in the culture that        retains the blue colour or shows no significant increase in        fluorescence compared with the control sample, is identified as        a strain of bacteriophage specific to a selected strain of        bacteria. A sterile sample that was treated with the same        incubation is used as a control sample,    -   f) identified strain of bacteriophage specific to a selected        strain of bacteria is propagated.

Favourably, the selected bacterial strain is a strain of S. entericaserovar Enteritidis.

The disclosed method is suitable for an easy and rapid screening oflarge collections of bacteriophage and allows for easy determination ofthe titre (lytic strength) of tested phages, which is essential inindustrial applications.

Another object of the invention is the use of bacteriophages tomanufacture a preparation for preventing and fighting infections of farmanimals, especially poultry, with pathogenic strains of bacteriasensitive to these bacteriophages. The manufactured preparation isintended to be administered to endangered animals with food or water, atintervals of one to seven days.

Favourably, the manufactured preparation provides at least 200-foldreduction in the level of infection a week after the administration isstopped.

Favourably, the infection to be fought is an infection of poultry withpathogenic strains of Salmonella sp., whereas in order to manufacturethe preparation, a bacteriophage strain is selected from the groupconsisting of those strains disclosed in this application, deposited on7 Jun. 2011, in the Polish Collection of Microorganisms, under thefollowing deposit numbers: PCM F/00069 (strain 8 sent 1748), PCM F/00070(strain 8 sent 65) and PCM F/00071 (strain 3 sent 1), is used.

Another object of the invention is a strain of bacteriophage suitable toprevent or fight infections by pathogenic strains of Salmonella sp.,selected from the group consisting of: PCM F/00069 (strain 8 sent 1748),PCM F/00070 (strain 8 sent 65) and PCM F/00071 (strain 3 sent 1).

The preparation according to the invention is based on naturalcomponents of the ecosystem and has no unfavourable influence onorganisms other than specific pathogenic bacteria. While commerciallyavailable substitutes of antibiotics are based on substances which, likefor example, organic acids, non-specifically modulate the bacterialflora, preventing to some extent the growth of undesirablemicroorganisms, the preparation according to the invention ensures thatonly pathogenic strains of Salmonella spp are selectively reduced.Unexpectedly, it also appeared that the preparation according to theinvention is not maintained in the human or animal body, if there is noSalmonella spp present. In a particular realization, the preparation issuitable for use in animal production, especially to fight Salmonellainfection in poultry.

Bacteriophage strains disclosed in this application were identified by aprocess according to the invention. Unexpectedly, they show a widespecificity involving the lysis of at least four specific serotypes ofSalmonella and maintain to be stable in refrigerated storage conditionsfor at least 3 months. Moreover, they can be successfully propagated onan industrial scale without loss of activity and they are not specificto the pro-biotic Lactobacillus bacteria.

In order to clarify the invention, it has been illustrated in theaccompanying figures which present:

FIG. 1: The restriction profiles for selected bacteriophages;

FIG. 2: The graphs of the monitored parameters of the conductedexperiments;

FIG. 3: Sample image of ELISA plates immediately after adding thereaction mixture and after three hours of incubation. The OD600 opticaldensity of the used suspension of S. enterica ser. Enteritidis ATCC13076 strain amounting to 0.05, 0.1, 0.25, 0.5, 1.0 and 2.0,respectively corresponds to the density of cells—8×105, 1.05×106,7.0×106, 3.5×107, 1.4×108 and 3.8×108;

FIG. 4: The results of thermal stability of selected bacteriophagescarried out within three months in three different temperatureconditions;

FIG. 5: Results of bacteriophage preparation's cytotoxicity testing withneutral red method. A preparation containing a sterile mixture ofbacteriophages was added for 24 h to culture of mouse 3T3 fibroblasts,and cell viability was assessed with the use of neutral red uptake test;

FIG. 6: Detection of Salmonella bacilli in the experiment which wascarried out;

FIG. 7: The level of CFU Salmonella in internal organs and intestines of21-day-old chicks.

The herein description has been supplemented with the following exampleswhich serve for better illustration of the nature of the invention.However, these examples should not be identified with the full scope ofthe invention.

EXAMPLE 1 Isolation and Description of Bacteriophages

Establishing a Bank of Salmonella Strains (Serovars) Most FrequentlyIsolated from Humans and Farm Animals

The set of 108 Salmonella ssp. strains, consisting of the mostfrequently isolated serovars, was collected for the necessity of theproject (Table 1). These strains are applied in determination ofspecificity of purified bacteriophages. The collection is comprised ofboth reference strains available in public repositories, as well asisolates obtained owing to cooperation with the VETLAB company (Brudzew,Poland) and State Sanitary Inspection.

TABLE 1 The list of Salmonella enterica serovars and their origins.Number of No. Serovar strains Source 1 Berta 1 VetLab 2 Brandenburg 1State Sanitary Inspection 3 Coeln 1 VetLab 4 Colindale 1 VetLab 5 Derby1 VetLab 6 Enteritidis 1 ATCC 1 State Sanitary Inspection 58 VetLab 7Gallimarum 1 VetLab Pullorum 8 Hadar 2 VetLab 1 State SanitaryInspection 9 Heidelberg 1 VetLab 10 Infantis 6 VetLab 1 State SanitaryInspection 11 Mbandaka 1 VetLab 12 Moscow 1 VetLab 13 Newport 5 VetLab14 Paratyphi 1 ATCC 15 Senfenberg 1 VetLab 16 Typhi 1 ATCC 17Typhimurium 1 ATCC 1 State Sanitary Inspection 10 VetLab 18 Virchow 9Vetlab

Isolation of Bacteriophages, from Environmental Samples, ShowingActivity Towards Selected Serovars of Reference Salmonella ssp Strains

Bacteriophages were isolated from samples provided by the WastewaterTreatment Plants from Lodz, Poland and Tuszyn, Poland. The studiesconfirmed that the samples collected from sands' separation stage (sandchamber), which is one of the wastewater's treatment processes, are therichest source of viruses. Moreover, the bacteriophages were alsoobtained from hen feces, provided by a private farmer and VETLAB companyspecializing in bacterial contamination analysis of farms. Isolation ofphages was carried out with the use of Salmonella enterica referencestrains, including Typhimurium, Enteritidis and Typhi serovars, as wellas several environmental strains. So far, several selected phages weredescribed in details (Table 2).

TABLE 2 The list of obtained bacteriophages and their host strains. No.Bacteriophage Source Host strain 1 1st1 Wastewater S. enterica ser.Typhimurium LT2 treatment plant 2 1sent3 Wastewater S. enterica serEnteritidis ATCC treatment plant 13076 3 1sent4 Wastewater treatmentplant 4 3sent1 Wastewater treatment plant 5 4sent1 Private farm 6 6sent1Private farm 7 5sent1 VetLab S. enterica ser. Enteritidis 65/S/10 88sent65 VetLab 9 8sent1748 VetLab S. enterica ser. Enteritidis 1748 102styp4 VetLab S. enterica ser. Typhi ATCC 13311 11 2styp5 VetLab 126styp1 Private farm

All phages intended for further investigations were purified with theuse of the screening method in order to obtain a single plaque on the LB(Luria-Bertani) plates. This procedure required at least five timeapplication of the screening process.

Initially, specificity of viruses isolated with the use of the platemethod was defined by determining the lytic capability of isolatedbacteriophages towards 17 selected S. enterica strains, including 7various serovars selected, 9 strains of Enteritidis serovars isolatedfrom humans and farming animals, as well as the host strains of analyzedbacteriophages (Table 3). In order to confirm the results, thespecificity determination of isolated bacteriophages was repeated threetimes.

TABLE 3 Specificity of selected bacteriophages in relation to chosenreference and environmental strains. S. enterica Bacteriophage serovars1st1 1sent3 1sent4 3sent1 4sent1 5sent1 6sent1 8sent65 8sent1748 2styp42styp5 6styp1 Typhimurium + − − + − + − + + − − + LT2 Typhimurium − − +− − − − + − − − − 1751 Typhi ATCC + − − + − + − + + + + + 13311Paratyphi A − − + − − − − + − − − + ATCC 19150 Infantis − − − − − − − −− − + − 789 Brandenburg + − + + − + − + + − − − 584 Hadar + − − + − −− + + − − − 817 Enteritidis D + + + + + + + + + − − + ATCC 13076Enteritidis + + + + − + − + + − − − 1748 Enteritidis + − + +− + + + + + + − 65/S/10 Enteritidis + + + + + + nd + + − − nd 249Enteritidis + + + + − + nd + + − − nd 1014/S/09 K-1 Enteritidis + − + +− + nd + − − − nd 1192/S/09 K-8 Enteritidis + − + + − + nd + + − − nd1250/S/09 Enteritidis + + + + − + nd + + − − nd 1446/S/09 K-31Enteritidis + + + + + + nd + + − − nd 1535/S/09 Enteritidis + + + + + +nd + + − − nd 2050/S/09 K-4 nd—not determined

The resulting phages, propagated in the host strain, were concentratedby PEG8000. Such prepared samples were subjected to the process ofisolation of genomic DNA of studied bacteriophages. This procedure useszircon spheres of 0.1 mm diameter. It also uses extraction with organicsolvents and commercial systems for the isolation of genomic DNA. Theresulting DNA is used in (1) restriction analysis (three independentexperiments using the EcoRI enzyme) generating various restrictionprofiles for different bacteriophages, which represents an initial genecharacterization of phages (FIG. 1).

A more detailed genetic characterization is obtained from sequencing thegenomes of bacteriophages from our collection, performed with the use ofnew-generation sequencing techniques, subcontracted to the Macrogencompany. Analysis of the results of such sequencing is performed by ourresearch team. It was established that so far obtained sequences of thegenomic DNA show high homology towards the bacteriophage from very wellexplored T5 family and are the lytic bacteriophages.

EXAMPLE 2 Manufacture of the Preparation

Determination and Optimization of Bacteriophage Propagation ProcessConditions in a Salmonella ssp. Culture at a Laboratory Scale

Optimization was carried out with the use of Salmonella enterica ser.Enteritidis ATCC 13076 strain. The parameters taken into considerationwere as follows: inoculum volume of a bacterial culture andbacteriophages, time of the pure culture process and incubation ofinfected culture, temperature of the culture, aeration level, pH ofmedium and conditions required for lytic cycle induction.

The optimal inoculum volume of bacterial culture was assessed to be2×10⁹ CFU for 0.5 l of culture medium. The optimized culture process wasbeing carried out until optical density reached the level of OD₆₀₀=0.5,which was achieved after 3 hours of incubation. The temperature of 37°C. appeared to be the optimal for bacterial growth. The optimal aerationlevel was achieved by shaking at 220 rpm in the New Brunswick shaker.The optimal culture's growth was observed on the LB medium of pH=7.0.The optimization process demonstrated that the addition of 10% ofbacteriophage suspension showing titer of 10⁹ (50 ml per 500 ml ofculture) is the most favourable inoculum amount. Also, the analysisshowed that the optimal initial proportion of bacteriophages particlesand bacterial cells is 25:1. Moreover, the optimization studies revealedthat the isolated bacteriophages display lytic nature and consequentlyinduction of the lytic cycle is not required.

The process of bacteriophage's recovery from culture was performed byultracentrifugation in Beckman L-80 type ultracentrifuge. Bacterialcultures infected with bacteriophages after proper incubation process(see above) were initially centrifuged for 30 min (3700 g). Afterwards,the supernatant was transferred into Beckman type ultracentrifugationtubes and ultracentrifuged for 2 hours (200 000 g). This procedureallowed for simultaneous purification and concentration of phagepreparation.

Optimization of Salmonella spp Reference Strain Culture in 10-LitersScale

In the next step of the optimization, bacteriophage propagation methodin a 10-liters bioreactor (8-liters of working volume) was developed.For this purpose, 8 litres of LB medium was prepared and autoclaved (for20 min. at 120° C.) in the bioreactor. Before inoculation with 200 ml ofSalmonella Enteritidis 65 strain, the medium was aerated to 90% -100%and heated up to 37° C. The inoculum was a 16-hour bacterial cultureshowing optical density of approximately OD₆₀₀=5.0 (4.5-5*10⁹ CFU/ml).After inoculation of the medium, sample was taken in order to measureoptical density (OD₆₀₀) of Salmonella Enteritidis 65 initial culture.The crucial parameters of the culture and the final titer of propagatedbacteriophages were monitored (Table 4 and 5). The culture was carriedout in bioreactor with constant aeration at 1.3 lpm (air volumedelivered to the medium during 1 minute) and stirring at 50 rpm. After40 minutes of the process, stirring intensity was increased from 50 to100 rpm. During the culture process, samples were taken every 30 minutesfor determination of optical density of the culture (OD₆₀₀). When thebacterial culture's optical density (OD₆₀₀) of the value of 0.48-0.55was achieved, a 3sent1 bacteriophage suspension of suitable titer wasadded into the culture, in the volumes as follows:

-   -   800 ml in case of experiment 1,    -   700 ml in case of experiment 2, as well as    -   200 ml in case of experiment 3 and 4.

From this point of time, the culture was carried out for 4 hours withconstant aeration and stirring (see above). The changes of opticaldensity and phage propagation kinetics were monitored by taking samplesevery hour. During the process, culture parameters, including pH, pO₂(aeration level indicated in %), temperature of the medium and correctlevel of stirring, were monitored. Every monitored parameter wasrecorded with the use of software designed for documentation of thecourse of culture process (FIG. 2), except experiment 1, due totechnical problems experienced with initializing of the software. Duringthe conducted experiments, progressing decrease of dissolved oxygenlevel in medium (increase of oxygen consumption by bacterial cells dueto their growth in the culture), as well as small pH fluctuations withinthe range of 7.2-6.7, were noted.

Regardless of the phage suspension volume used (from 200 to 800 ml withtiter of 1.3-2.3×10⁹ PFU/ml), high propagated bacteriophage titer ofabout 10⁹ PFU/ml was achieved.

TABLE 5 Parameters of 3sent1 phage's propagation in 10 liters ofSalmonella Enteritidis 65 strain culture. Volumes applied [ml]Experiment 1 Experiment 2 Experiment 3 Experiment 4 Bacterial inoculum200 ml 200 ml 200 ml 200 ml Phage suspension 800 ml 700 ml 200 ml 200 mlOD₆₀₀ Beginning of the 0.097 0.089 0.070 0.070 process Before addition0.488 0.501 0.501 0.535 of phage suspension 1h of phage's 0.589 0.7200.856 0.477 propagation 2h of phage's 0.150 0.286 0.632 0.198propagation 3h of phage's 0.110 0.210 0.372 0.160 propagation 4h ofphage's 0.068 0.182 0.320 0.166 propagation

TABLE 5 Number of propagated 3sent1 phage particles. Density of phagesuspension [PFU/ml] Experiment 1 Experiment 2 Experiment 3 Experiment 4Initial phage 2.32 × 10⁹ 1.17 × 10⁹ 1.33 × 10⁹ 1.44 × 10⁹ suspensionPFU/ml PFU/ml PFU/ml PFU/ml 1h of phage's 6.20 × 10⁹ 1.05 × 10⁹ 1.42 ×10⁹ 4.94 × 10⁹ propagation PFU/ml PFU/ml PFU/ml PFU/ml 2h of phage's5.54 × 10⁹ 4.32 × 10⁹ 5.98 × 10⁹ 3.90 × 10⁹ propagation PFU/ml PFU/mlPFU/ml PFU/ml 3h of phage's 6.26 × 10⁹ 3.84 × 10⁹ 5.72 × 10⁹ 5.80 × 10⁹propagation PFU/ml PFU/ml PFU/ml PFU/ml 4h of phage's 7.94 × 10⁹ 4.16 ×10⁹ 4.26 × 10⁹ 3.16 × 10⁹ propagation PFU/ml PFU/ml PFU/ml PFU/ml

After 4 hours of bacteriophage propagation process, the whole content ofbioreactor was centrifuged for 30 minutes at 4° C., at the speed of 4500rpm/min. Since the technical parameters applied are not effective enoughfor precise separation of biomass from medium; at the next step thesupernatant containing phage particles was micro-filtrated twice withthe use of the filtration cross-flow system with membrane cassettes forseparation of remaining biomass and sterilization of the obtained phagepreparation

CONCLUSIONS: The performed biotechnological processes enabled us todescribe initial restrictions for phage particles propagation methodusing Salmonella spp strain culture in 10-liters bioreactor scale.

-   -   Volume of medium—8 litres;    -   Volume of Salmonella Enteritidis 65 inoculum (OD₆₀₀=5; 4.5-5×10⁹        CFU/ml)—200 ml;    -   Volume of phage suspension (titer 10⁹ PFU/ml)—200 ml;    -   Time of phage particles propagation process—3 hours;    -   Parameters of centrifugation—30 minutes at 4° C., at the speed        of 4500 rpm/min;    -   Two-time microfiltration (membrane with pore size of 0.22 μm);    -   Titration procedure of the obtained phage preparation;

Development of the Preparation's Manufacturing and PurificationTechnology

Stages of Bacteriophage Preparation's Manufacturing Process

1. Bioreactor Culture.

Propagation process of bacteriophage particles is the first stage of theproduction line. This is realized by inoculating a bacterial culture ofSalmonella in a bioreactor (conditions described above) withbacteriophage particle suspension. During the culture process, phageparticles are propagated in bacterial cells leading to cell lysis. Eachof all three bacteriophages has to be propagated in a separate culture.So far in our research, a classic 10-liter bioreactor (8 litres ofworking volume) has been used. In order to carry out this type ofculture, previously prepared components are used, including (1)bacterial inoculum and (2) bacteriophage suspension which is added afterachieving a suitable optical density (OD₆₀₀) of the bacterial culture.After addition of phage suspension, the culture is carried out from 3 to4 hours and the process enables to obtain a high titer of a propagatedbacteriophage at the level of about 10⁹ PFU/ml. Once the propagationprocedure is finished, the culture is transferred in a sterile manner tothe next stage of production process by means of peristaltic pump. Inthe future, 100-liter bioreactors or single-use advanced bioreactor bags(up to 15 litres), which lately successfully replace classicbioreactors, are planned to be applied at the production line.

2. Removal of Biomass.

Since the total lysis of bacterial culture during the bioreactorincubation process is not possible, a subsequent stage separation of thebiomass from phage-containing culture liquid is required. Therefore, atthe first stage, the culture is transferred to a centrifuge, and thenmicrofiltration process is carried out twice using cross-flow filtrationsystem containing membrane with pore size of 0.22 μm. This procedureallows to obtain a sterile bacteriophage suspension with very smalldecrease in phage particles titer. Once the filtration process isfinished, the suspension is transferred in a sterile manner to the nextstage of production process by means of peristaltic pump. In the future,expansion of this stage of the production line by purchasing additionalfiltration systems is planned. This would allow for treatment of thebiological material obtained from a single bioreactor culture withoutthe need for cleaning and sterilization of the filtration equipment usedduring this stage.

3. Product Concentration (Optional).

Depending on the product demand, bacteriophage suspension can beconcentrated 10-fold which allows for increase of the number of phageparticles in a given volume. Therefore, a cross-flow filtration systemwith ultra-filtration membrane of 30 or 50 kD cut-off (depending on thephage), is used. This process allows for 7.5-fold concentration ofbacteriophage particles.

4. Removal of Potential Endotoxin Contamination.

In order to eliminate endotoxin remaining after lysis of bacterial cellsinduced by bacteriophages during the biotechnological process,application of special adsorption columns is planned. These columns willcontain resin suitable for removing endotoxin of size and capacitydepending on the volume of the phage-containing preparation.

5. Preparation of the Product in Liquid Phase.

At this stage, a mixture of different bacteriophage suspensions,obtained in procedures described above, is prepared. The mixture shouldcontain all bacteriophages with very similar titer value. Bacteriophagesselected for the preparation show lysis capability of widest spectrum ofSalmonella spp bacterial strains from the possessed collection.Subsequently, the preparation is portioned in strictly sterileconditions. Additionally, during this process the final suspension isagain subjected to sterilization by means of a single-use, micro-filters(0.22 μm). 6. Microencapsulation (Optional)

Depending on whether the product is required in liquid or solid form,implementation of microencapsulation technology is planned in order toenclose bacteriophage particles in alginate capsules. The purpose ofthis stage is to generate easily absorbable capsules which safely passthrough the digestive system and to release gradually the enclosedbacteriophage particles in the target place without irritating the wholeorganism.

EXAMPLE 3 Research of the Effectiveness and Safety of the Preparation InVitro Research

Development and Optimization of the Highly Efficient and AutomatedColorimetric Method for Measurement of Bacteriophage PreparationActivity

The commercially available alamarBlue® reagent was used for developmentof the assay useful for measuring the bacteriophage lytic activity. ThealamarBlue® is an indicatory dye which enables fast and accuratequantification of proliferation and cytotoxicity based on chromaticreaction. An easy-to-use reagent takes advantage of the phenomenon of areduction-oxidation reaction (REDOX) and during the process a resazurinchanges its colour from blue (non-fluorescent) to red (fluorescent) as aresult of oxidation, which is a consequence of cell metabolic activity.The change of colour is visible with the naked eye. It can be alsomeasured spectrophotometrically or fluorometrically. Due to its lack ofcell toxicity, alamarBlue® is especially valuable in observation ofbacterial cell differentiation in microbial cultures. This test wasadjusted for application on 96-well ELISA plate and two techniques ofmeasurement were elaborated. The first one was intended to assess thelytic activity of bacteriophages. During the test, the so called freshworking mixture was used (prepared just before application on the ELISAplate), which consisted of alamarBlue® and 20% sterile Tween™ 80solution (3 parts of alamarBlue° and 1 part of 20% sterile Tween™ 80solution), and was added to wells containing studied Salmonella strainsuspension of appropriate density, as well as to wells filled with bothSalmonella cell suspension of appropriate density and studiedbacteriophage suspension of desired titer. The procedure enables visualdetermination of the lytic activity of phage particles in relation tothe investigated Salmonella strains. The blue colour of the reactionmixture of alamarBlue®+20% sterile Tween™ 80 solution (3 parts ofalamarBlue® and 1 part of 20% sterile Tween™ 80 solution) indicates lackof growth of the studied Salmonella strain and high lytic effect ofproper bacteriophage concentration. Colour change of the reactionmixture of alamarBlue®+20% sterile Tween™ 80 solution (3 parts ofalamarBlue® and 1 part of 20% sterile Tween™ 80 solution) from blue tored, caused by the oxidation associated with metabolic activity,indicates growth of Salmonella cells and lack or low lytic effect ofproper bacteriophage concentration in relation to the investigatedSalmonella strain.

A detailed experimental protocol enabling quantitative assessment of thelytic activity of bacteriophages, as well as a drawing presenting theresults of a selected experiment conducted for the 3sent1 bacteriophagein relation to Salmonella enterica ser. Enteritidis ATCC 13076 strain(FIG. 3), are presented below.

-   -   1. Add 100 μl of Salmonella spp. culture suspension of proper        optical density (OD₆₀₀) relating to appropriate number of colony        forming units (CFU/1 ml)¹ into each well of the first row in        columns from 1 to 12. In detail: suspension of the optical        density of 0.05, 0.1, 0.25, 0.5, 1.0 and 2.0 into the plate        wells: 1 and 7, 2 and 8, 3 and 9, 4 and 10, 5 and 11, as well as        6 and 12, respectively.

¹Relation between the OD₆₀₀ optical density and the CFU value should bedetermined experimentally in several independent approaches.

-   -   2. Add 100 μl of phage suspension at concentration of 6.5×10⁹        PFU (plaque forming units) into each well of the first row in        columns from 1 to 6. The volume proportion of bacterial to phage        suspension is 1:1.    -   3. Similarly, add 75 μl of Salmonella spp. culture suspension of        proper optical density and 125 μl of phage suspension into wells        of the second row. The volume proportion of bacterial to phage        suspension is 1:1.5.    -   4. Similarly, add 50 μl of Salmonella spp. culture suspension of        proper optical density and 150 μl of phage suspension into wells        of the third row. The volume proportion of bacterial to phage        suspension is 1:3.    -   5. Prepare the controls; add sterile LB medium instead of phage        suspension into each well of the first, second and third row in        columns from 7 to 12 (control of Salmonella strain growth of        given densities). Additionally, add phage suspension into each        well of 6^(th), 7^(th) and 8^(th) row in column 3 (control of        phage suspension contamination) and add LB medium into each well        of 7^(th) and 8^(th) row in column 7 (control of contamination        of the medium applied to dilute the culture in investigated and        control set).    -   6. Cover the ELISA plate with sterile lids and incubate for 4 h        at 37° C. After the incubation stage, add 50 μl of        alamarBlue®+20% sterile Tween™ 80 reaction mixture² into each        well plate. Due to the reaction mixture's susceptibility to        light, protect the plate with aluminium foil. Continue the        incubation for the next 3 h in 37° C. Perform visual observation        every 30 minutes. ²The mixture should be prepared directly        before application into wells on ELISA plate (3 parts of        alamarBlue® and 1 part of 20% sterile Tween™ 80 solution).

The modified protocol may be applied as a useful tool for estimation ofprotective effect of bacteriophages against Salmonella Enteritidisgrowth. Colour change of the reaction mixture of alamarBlue® from blueto red, which is caused by the oxidation associated with metabolicactivity, indicates Salmonella cells’ growth. The blue colour of thereaction mixture of alamarBlue®+20% sterile Tween™ 80 solution indicatesprotective effect of phage suspension preventing growth of Salmonellacells. The detailed protocol:

-   -   1. Add 20 ml of LB medium to 100 ml Erlenmayer conical,        flat-bottom flasks and autoclave.    -   2. Inoculate 20 ml of sterile LB liquid medium with S.        Enteritidis strain and incubate the culture overnight at 37° C.        with shaking (rpm=150). The suspension shall be stored in        refrigerator at 4° C. not longer than for 1 week and treated as        inoculum for current cultures.    -   3. Inoculate 20 ml sterile LB liquid medium (in 100 ml        Erlenmayer flasks) with 20 μl of S. Enteritidis strain and        incubate the culture for 1 h at 37° C. with shaking (rpm=150).    -   4. Make appropriate dilutions of the suspension in LB. Dilutions        should contain 2000, 200 and 20 bacterial cells in 1 ml. Use for        experiments with phage suspension showing protective effect.    -   5. Mix 100 μl of the appropriate dilution of S. Enteritidis        bacterial cells with 100 μl of bacteriophage suspension and add        the mixtures into the sequential wells of 96-wells titer plate.    -   6. Prepare the controls; appropriate dilutions of S. Enteritidis        suspension which will serve as bacterial growth control, as well        as the bacteriophage suspension and a clear LB medium as the        controls of microbial contamination.    -   7. Cover the titer plate with a lid and incubate for 4 h at 37°        C.    -   8. Cover the titer plate with a lid and protect with aluminium        foil against admission of light, then continue the incubation        for next 4 h at 37° C. Perform the reading, after 4 hours of the        incubation process.

Research of Thermal Stability of Bacteriophages

Analysis of thermal stability of bacteriophages storage was carried outfor 3 months at three different temperatures: −20° C., +4° C. and atroom temperature. The stability was measured by determining phage titerby means of plate count at the beginning of the experiment, after onemonth and after three months of storage in the mentioned abovetemperatures. Bacteriophages were suspended in LB medium afterpropagation and purification from bacterial cells. The results indicatethat some of bacteriophages of the existing collection, namely 3sent1,8sent65 and 8sent1748 (FIG. 4), retain their title at the order ofmagnitude within three months of storage at −20° C. or 4° C., contraryto other phages, e.g.: 1st1 or 2styp5 (FIG. 4), which is important inview of durability.

Research of Bacteriophage Preparation's Safety

Determination of Endotoxin Level

Determination of endotoxin concentration (LPS), the major component ofcell's wall of gram negative bacteria, was performed using LAL test(Limulus Amebocyte Lysate). The LAL tests are available as ready-to-usekits for colorimetric measurement of LPS level and activity:

-   -   1. Kit produced by Genscript (ToxinSensor LAL Endotoxin Assay        Kit).    -   2. Kit produced by LONZA (QCL-1000 Endpoint chromogenic LAL        Assay).

In these tests, LAL reagent interacts with endotoxin in analyzed samplesresulting in production of a reaction product which is able to reactwith chromogenic substrate, thus the spectrophotometric measurement ispossible. The intensity of the colour is directly proportional toendotoxin concentration.

Phage-containing samples were taken at different stages of thetechnological process (assessment of ultracentrifugation'seffectiveness—A1-A2 samples, and phage particles concentration usingdifferent membranes cut-off: 100 kDa—B1-B5 samples and 50 kDa—C1-C7samples) and analyzed for endotoxin level. The results obtained indicatepresence of high amount of LPS in samples taken after the phageparticles concentration stage (Table 6).

TABLE 6 Results of LPS concentration in analyzed samples. LPS Sampleconcentration No. Description EU/ml A1 Sample containing pellets(phages) after ultracentrufugation >10⁶ suspended in SM buffer A2 Samplecontaning supernatant after ultracentrifugation 106 345 B1 Initialsample —medium contaning phages after seperation of bacterial >10⁶ cellsand sterilization B2 10-fold concentrated preparation (retentate) 156389 B3 Permeate—liquid filtered through a concentration membrane 4 888B4 Saline used to flush the membrane after the end of filtrationprocess, 55 collected as permeate B5 Saline used to flush the membraneafter the end of filtration process, 0.5 collected as retentate C1Initial sample—medium contaning phages after seperation of bacterial 211327 cells and sterilization C2 Permeate—liquid filtered through aconcentration membrane 25 C3 10-fold concentrated preparation(retentate) 1.36 × 10¹⁰ C4 Saline used to flush the membrane after theend of filtration process, 1.69 × 10⁹  collected as retentate (firstsample 100 ml) C5 Saline used to flush the membrane after the end offiltration process, 1.32 × 10⁹  collected as retentate (second sample200 ml) C6 Saline used to flush the membrane after the end of filtrationprocess, 1.05 × 10⁸  collected as retentate (third sample 200 ml) C7Saline used to flush the membrane after the end of the filtration 1 549process, collected as retentate (fourth sample 500 ml)

Cytotoxicity Analysis

Investigation was carried out by means of the neutral red uptake assay.The test was performed with 96-wells plates using 3T3 cell line. 3T3cells are mouse-derived, not-transformed fibroblasts conventionally usedfor in vitro toxicity analysis. Analysis was carried out on thepreparation containing a mixture of three different phages in 4 serialdilutions. Measurements were performed in duplicates and in 5independent repetitions. Obtained absorbance values were used forcalculation of % of cells' viability by comparing the absorbance ofanalyzed sample with absorbance of control sample (i.e. cells incubatedin culture medium) (FIG. 5).

Cytotoxicity test using 3T3 cell line:

-   -   1. Put 10 000 3T3 fibroblasts suspended in standard culture        medium to each well of 96-wells plate and incubate for 24 hours        in the incubator (37° C.; 5% CO₂).    -   2. Remove culture medium and add bacteriophage preparation        diluted in culture medium. Continue the incubation for next 24        hours (37° C.; 5% CO₂).    -   3. After the incubation, remove medium containing phage        preparation, wash the monolayer of PBS fibroblasts and incubate        with neutral red solution in PBS for 3 hours (37° C.; 5% CO₂).    -   4. Remove neutral red solution, wash with PBS and induce cell        lysis. Measure the amount of absorbed dye by colorimetric        method.

Obtained results indicate lack of cytotoxic activity of bacteriophagepreparation even at high concentrations. Consequently, acutecytotoxicity is not expected to appear after using this preparation invivo at phage concentration of at least up to 10⁸ particles.

In Vivo Research

Determination of Effectiveness and Safety of Prototype BacteriophagePreparation Applied Against Salmonellosis in Chicken Broilers.

The study was carried out in cooperation with the University of Warmiaand Mazury, as well as VETLAB.

Purpose: To evaluate the possibility of using bacteriophages inprotection against salmonellosis infections in broiler chickens.

Salmonella enterica ser. Enteritidis 65/S/10 strain provided by VETLABwas used to infect broilers, whereas to prepare the bacteriophage agent,three bacteriophages: 3sent1 , 8sent65, and 8sent1748, isolated from twoSalmonella enterica strains (Table 3) and showing a wide spectrum ofspecificity against different Salmonella serovars, as well as againstinvestigated Enteritidis strains, were used. Based on specificities ofthe used bacteriophages, specificity of the complete preparation can beassumed (Table 7). Bacteriophage preparation was prepared in thefollowing manner: each of the three bacteriophages were subjected to theoptimized procedure of propagation, and afterwards the obtained phagesuspensions were mixed, so that their titer values in the final productwere similar. Two mixtures with different phage concentrations wereprepared: high concentration containing 2.0×10⁸ PFU/ml and lowconcentration containing 2.0×10⁶ PFU/ml. Afterwards, the mixtures wereportioned and sterilized using microfiltration. Analysis of microbialcontamination showed no presence of bacteria in the bacteriophagepreparation used.

TABLE 7 Assumed specificity of the preparation based on specificity ofphages included. PREPA- Serovar Strain 3sent1 8sent1748 8sent65 RATIONTyphimurium LT2 + + + + 1751 − + − Typhi ATCC 13311 + + + + Paratyphi AATCC 19150 − + − Infantis 789 − − − Brandenburg 584 + + + + Hadar817 + + + + Enteritidis D ATCC 13076 + + + + 1748 + + + +65/S/10 + + + + 249 + + + + 1014/S/09 K-1 + + + + 1192/S/09 K-8 + + −1250/S/09 + + + + 1446/S/09 K-31 + + + + 1535/S/09 + + + + 2050/S/09K-4 + + + + Farm “K” K-3 + + + + Farm “K” K-6 + + + + Farm “K” K-9 + − −Farm “K” K-10 + + − Farm “K” K-11 + − − Farm “K” K-12 + − − Farm “K”K-14 + + + + Farm “K” K-18 − − − Pac K-ground − + + Pac K-floor + + + +W/K-6 + + + + W/K-7 + + − W/K-9 + + + + 64/S/10 + + + + 65/S/10 + + + +517/S/09 + + + + 571/S/09 + + + + 833/S/09 + + + + 838/S/09 + − +838/S/09 B + + + + 847/S/09 + + + + 848/S/09 + − − 865/S/09 + + + +866/S/09 + + + + 945/S/09 + + + + 975/S/09 + + + + 1013/S/09 K-4 + + + +1021/S/09 + + + + 1022/S/09 K-9 + + + + 1044/S/09 K-2 + − + 1047/S/09K-1 + + + + 1048/S/09 K-8 + + + + 1061/S/09 K-5 − − − 1067/S/09 K-2 + −− 1067/S/09 K-3 + + + + 1085/S/09 MEK + + + + 1106/S/09 K-1 + + + +1143/S/09 + + + + 1171/S/09 + + + + 1206/S/09 MEK + + + +1231/S/09 + + + + 1250/S/09 + + + + 1257/S/09 K-7 + + + + 1422/S/09MEK + + + + 1445/S/09 K-46 + − + 1465/S/09 MEK + + + + 1535/S/09 + + + +1542/S/09 NWJ + + + + 1545/S/09 MEK + − + 1572/S/09 NWJ + + + +1573/S/09 NWJ + + + + 1714/09 + + + + 1748 + + + + 2149/09 + + + +2619/S/10 + + + +

Experimental Procedure

One hundred fifty Ross 308 roosters, divided randomly into 5 equal,isolated groups (separate rooms—boxes), were used for the research.Chickens were fed with standard, complete feed produced by AgrocentrumKolno, Poland and kept in conditions complying with recommendation ofthe biological material's manufacturer. Four-days-old chickens fromboxes 1, 2 and 3 were infected with Salmonella Enteritidis bacilli in adose of 1×10⁵ CFU per animal (Table 8). Chickens from boxes 4 and 5 werenot infected with Salmonella bacilli. The bacteriophage preparation wasadministered to chickens placed in boxes 1 (low concentration of 2.0×10⁶PFU/ml, marked as F_(N)), 2 and 5 (high concentration of 2.0×10⁸ PFU/ml,marked as F_(W)). The preparation was administered to roosters once aday for first 14 days of their lives. The bacteriophage agent was notadministered to chickens from boxes 4 and 5. The rearing time was 21days and during this period only five birds from different boxes died(Table 8). Since the detected death rate was minimal and did not dependon a given group of chickens therefore the death rate could not havebeen the result of the administration of the phage preparation.

TABLE 8 The scheme of administration of bacteria and bacteriophages, aswell as birds' viability. Salmonella Bacteriophage Chicken's Boxinfection administration viability 1 1 × 10⁵ CFU/bird 2.0 × 10⁶ PFU/ml30/30 2 1 × 10⁵ CFU/bird 2.0 × 10⁸ PFU/ml 29/30 3 1 × 10⁵ CFU/bird —28/30 4 — — 30/30 5 — 2.0 × 10⁸ PFU/ml 28/30

Detection of Salmonella Bacilli

Samples for Salmonella bacilli detection were collected according to thescheme described below (FIG. 6):

-   -   Investigation of feces during 21 days of experiment,    -   Investigation of litter—sole smears were collected on 3^(rd),        15^(th) and 21^(st) day of birds' life,    -   Investigation of liver, spleen and intestines of 21-days-old        broilers,    -   Investigation of empty boxes before insertion of        nestlings—smears were collected from walls, feeders, drinkers        and floor (control),    -   Investigation of one-day-old nestlings—internal organs,        intestines and meconium (control).

Detection of Salmonella bacilli's presence in analyzed samples wascarried out by an accredited laboratory—VETLAB (Brudzew, Poland). In2008, this laboratory received authorization of the Chief VeterinaryOfficer (no. GIWhig-5120-23/08) to perform detection of Salmonellabacilli by means of bacteriological-qualitative method.

Salmonella bacilli were not detected in analyzed samples of internalorgans and intestines of one-day-old nestlings, meconium, as well assmears from boxes before insertion of nestlings. The lack of Salmonellabacilli or different level of its detection in case of analysis of fecesfrom 21 days of experiment, as well as sole smears analysis on 3^(rd),15^(th) and 21^(st) day of life (Table 9), and internal organs analysisof 21-days-old broilers (FIG. 7) was dependent on the box underinvestigation. That is, as it was anticipated, no Salmonella bacilliwere detected in chickens from boxes 4 and 5. In case of birds from box3, which were infected with Salmonella bacilli and not treated with thebacteriophage preparation, the bacilli were detected starting from sixthday of life. Whereas, in case of chickens from boxes 1 and 2, which wereinfected with Salmonella bacilli and treated with the phage agent,inhibition of bacteria propagation was clearly seen until administrationof the phage preparation was stopped. What is important, the number ofbacilli detected in internal organs and intestines of 21-days-old birdswas very low in relation to chickens which were not treated with thephage preparation. Thus, bacteriophages prevent propagation of bacteriaand consequently the appearance of bacteria in feces of birds infectedduring the experiment. Accordingly, it can be observed thatbacteriophages decrease 200-fold the infection level even a week afterdiscontinuation of phage treatment.

TABLE 9 Level of Salmonella bacilli's occurrence in feces during theexperiment. Salmonella bacilli occurrence Box 1 Box 2 Box 3 Box 4 Box 5Phage Day Infection F_(N)+/S+ F_(W)+/S+ F−/S+ F−/S− F_(W)+/S− addition 1− − − − − Yes 2 − − − − − Yes 3 2.5 × 10³ − − − − − Yes 4 − − − − − Yes5 − − − − Yes 6 − − 1.0 × 10¹ − − Yes 7 − − + − − Yes 8 − − 5.3 × 10³ −− Yes 9 − − + − − Yes 10 − − 1.0 × 10⁴ − − Yes 11 − − + − − Yes 12 − 3.0× 10¹ 3.5 × 10⁴ − -Yes 13 − − + − − Yes 14 − 2.0 × 10³ 5.5 × 10³ − − Yes15 4.1 × 10³ 1.0 × 10² 1.5 × 10³ − − No 16 + + + − − No 17 2.1 × 10² +3.0 × 10⁴ − − No 21 5.0 × 10² 2.2 × 10³ 4.3 × 10⁴ − − No ″−″ means, noSalmonella bacilli were detected ″+″ means that Salmonella bacilli weredetected, but it was difficult to determine their titer

Detection of Bacteriophages' Occurrence.

Samples analyzed for bacteriophage presence were collected from fecesduring 21 days of the experiment and from smears from all boxes beforeintroduction of nestlings, before infection and also a day and a weekafter discontinuation of phage treatment. No bacteriophages weredetected in internal organs. It was observed that lack of bacteriophagesor different level of their detection in birds' feces (Table 10) andsole smears analyses (Table 11) depended on the given box of chickenunder analysis. That is, bacteriophages were detected in feces ofchickens form boxes 1, 2 and 5, which were administered the phage agent.However, their number decreased after discontinuation of the treatment,and especially in case of birds from boxes 2 and 5, where high phageconcentration was used, bacteriophages were no longer detected. It isworth adding that much smaller number of bacteriophages was detected infeces from box 5 compared to feces from box 2, which can be the resultof impossibility of phage's propagation due to the lack of Salmonellabacilli.

TABLE 10 Bacteriophage's titer level in poultry feces. Bacteriophage'stiter Box 1 Box 2 Box 3 Box 4 Box 5 Day FN+/S+ FW+/S+ F−/S+ F−/S− F+/S−1 0 0 0 0 0 3 0 1.8 × 10⁷ 0 0 5.6 × 10² 10 8.0 × 10⁶ 5.5 × 10⁴ 0 0 5.0 ×10³ 15 1.2 × 10⁷ 5.0 × 10¹ 0 0 8.2 × 10² 17 1.7 × 10⁴ 0 0 0 0 21 5.0 ×10¹ 0 0 0 0

TABLE 11 Bacteriophage's titer level in sole smears under analysis.Bacteriophage's titer Box 1 Box 2 Box 3 Box 4 Box 5 Day FN+/S+ FW+/S+F−/S+ F−/S− F+/S− One day after 1.0 × 10⁵ 1.0 × 10³ 0 0 2.4 × 10³discontinuation of phage treatment (15) One week after 6.5 × 10⁵ 0 0 0 0discontinuation of phage treatment (21)

Conclusions from the Investigation of the Effect of BacteriophagePreparation on Broilers

Based on the tests performed, one can conclude that the preparation issafe and efficient.

-   -   1. The preparation is safe for poultry.    -   2. Bacteriophages pass through the intestinal tract and reach        the feces and litter.    -   3. Bacteriophages do not enter internal organs of the birds.    -   4. Bacteriophages disappear after discontinuation of        administration of the preparation.    -   5. Bacteriophages prevent the appearance of bacteria in feces of        experimentally infected birds.    -   6. Bacteriophages decrease 200-fold the infection level one week        after discontinuation of the phage treatment.

1. The method of obtaining a strain of bacteriophage specific to aselected strain of bacteria, characterized in that: a) a collection ofbacteriophage strains containing a strain of bacteriophage specific to aselected strain of bacteria is obtained, b) culture of a selected strainof bacteria on a sterile culture medium is conducted, c) the culturesamples are applied on the special multi-well measuring plate, then asuspension of tested bacteriophage strain at various concentrations isadded and it is incubated at 37° C. for at least 4 hours, d) resazurinis added to the culture samples and incubation is continued in the darkat about 37° C. for at least 3 hours, e) the colour or fluorescence ofculture is being controlled, as well as bacteripohage strain containedin the culture that retains the blue colour or shows no significantincrease in fluorescence compared with the control sample, is identifiedas a strain of bacteriophage specific to a selected strain of bacteria.A sterile sample that was treated with the same incubation is used as acontrol sample. f) identified strain of bacteriophage specific to aselected strain of bacteria is propagated.
 2. Method according to claim1, characterized in that the selected bacterial strain is a strain of S.enterica serovar Enteritidis.
 3. Application of bacteriophages inmanufacturing of the preparation for preventing or fighting infectionsof farm animals, especially poultry, with pathogenic strains of bacteriasensitive to these phages, while the manufactured product is intended tobe administered with food or water to vulnerable animals at intervals ofone to seven days.
 4. Method according to claim 3, characterized in thatthe manufactured preparation provides at least 200-fold reduction in thelevel of infection for a week after discontinuation of administration.5. Method according to claim 3, characterized in that the infection tobe fought is an infection of poultry with pathogenic strains ofSalmonella sp, and in the preparation's manufacturing process,bacteriophage strain selected from the group consisting of: PCM F/00069(strain 8 sent in 1748), PCM F/00070 (strain 8 sent 65) and PCM F/00071(strain 3 sent 1) is used.
 6. Bacteriophage strain suitable for theprevention or eradication of infection by pathogenic strains ofSalmonella sp selected from the group consisting of: PCM F/00069 (strain8 sent in 1748), PCM F/00070 (strain 8 sent 65) and PCM F/00071 (strain3 sent 1) and exhibiting stability in refrigerated storage conditionsfor at least 3 months.